Chimeric canine anti-cd20 antibody

ABSTRACT

The disclosure relates to caninized chimeric anti-CD20 antibodies to the canine protein CD20 and methods of use to treat certain disorders such as non-Hodgkins B-cell lymphoma in dogs.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 10, 2016, isnamed “P20914 sequence listing” and is 14 Kb in size.

The present invention relates to caninized chimeric anti-CD20 antibodiesto the canine protein CD20 and methods of use to treat certain disorderssuch as non-Hodgkins B-cell lymphoma in dogs.

The present invention is in the field of treatment of cancer.

Canine lymphomas are a diverse group of cancers, and are among the mostcommon cancers diagnosed in dogs. They collectively representapproximately 7-14% of all cancers diagnosed in dogs. There are over 30described types of canine lymphoma, and these cancers vary tremendouslyin their behavior. Some progress rapidly and are acutelylife-threatening without treatment, while others progress very slowlyand are managed as chronic, indolent diseases. Lymphomas may affect anyorgan in the body, but most commonly originate in lymph nodes, beforespreading to other organs such as the spleen, liver, and hone marrow.

CD20 is a cell-surface protein with four transmembrane spanning regions.The CD20 molecule is involved in regulation of B-cell proliferation anddifferentiation. The CD20 antigen is present exclusively on the surfaceof almost all B-cells, both normal and malignant.

Rituximab is a chimeric monoclonal antibody against the human proteinCD20 which is primarily found on the surface of immune system B-cells.Rituximab destroys B-cells and is therefore used to treat diseases inhumans which are characterized by excessive numbers of B-cells,overactive B-cells, or dysfunctional B-cells. This includes manylymphomas, leukemias, transplant rejection, and autoimmune disorders.

Rituximab destroys both normal and malignant human B-cells that haveCD20 on their surfaces. The effect results in the elimination of B-cells(including the cancerous ones) from the body, allowing a new populationof healthy B-cells to develop from phoid stem cells.

Unfortunately, a cure for canine lymphomas still remains elusive andthere exists a need for more and different therapies that may prove tobe effective in treating them.

K9L0-133 is a chimeric anti-CD20 antibody that targets matureB-lymphocytes in dogs. K9L0-133 binds to CD2( )on canine B-lymphocytes.K9L0-133 is potentially useful for treatment for non-Hodgkins B celllymphoma and other types of lymphoma in dogs.

Accordingly, the present invention provides an antibody thatspecifically binds to canine CD20. The present invention also provides amethod of treating lymphoma in a canine patient by administering to alymphoma cancer canine patient in need of such treatment an effectiveamount of an antibody that specifically binds to canine CD20. Thepresent invention provides for a pharmaceutical composition including anantibody that specifically binds to canine CD20, and one or morepharmaceutically acceptable carriers, diluents, or excipients. Thepresent invention also provides an antibody that specifically binds tocanine CD20 and also binds to protein A.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts the serum levels of B-lymphocytes CD2 I+ and CD22+ andT-lymphocytes CD4+ and CD8+ from −5 to 37 days in dogs treated withK9LO-133 antibody.

DETAILED DESCRIPTION

Unless indicated otherwise, the term “antibody” (Ab) refers to animmunoglobulin molecule comprising two heavy chains (HC) and two lightchains (LC) interconnected by disulfide bonds. The amino terminalportion of each chain includes a variable region of about 100 to about110 amino acids primarily responsible for antigen recognition via thecomplementarity determining regions (CDRst contained therein. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

As used herein, the term “antigen-binding fragment” refers to anyantibody fragment that retains the ability to bind to its antigen. Such“antigen-binding fragments” can be selected from the group consisting ofFv, scFv, Fab, F(ab′)₂, Fab′, scFv-Fc fragments and diabodies. Anantigen-binding fragment of an antibody will typically comprise at leastone variable region. Preferably, an antigen-binding fragment comprises aheavy chain variable region (HCVR) and a light chain variable region(LCVR). More preferably, an antigen-binding fragment as used hereincomprises a HCVR and a LC\IR which confers antigen-binding specificityto canine CD20( )(i.e., a “canine CD20 binding fragment”).

As used herein, the terms “complementarity determining region” and“CDR”, refer to the non-contiguous antigen combining sites found withinthe variable region of LC and HC polypeptides of an antibody or anantigen-binding fragment thereof. These particular regions have beendescribed by others including Kabat, et al., Ann. N.Y. Acad. Sci,190:382-93(1971); Kabat et al, J. Biol. Chem, 252:6609-6616 (1977);Kabat, et al, Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo, 91-3242 (1991); Chothia, et al, J. Mol. Biol. 196:901-917 (1987);MacCallum, et al., J. Mol. Biol., 262:732-745 (1996); and North, et al.,J. Mol. Biol., 406, 228-256 (2011), where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother.

As used herein, the term “light chain variable region” refers to aportion of a LC of an antibody molecule that includes amino acidsequences of Complementarity Determining Regions (CDRs; i.e., LCDR1,LCDR2, and LCDR3), and Light Framework Regions (LERWs).

As used herein, the term “heavy chain variable region (HCNIR)” refers toa portion of a RC of an antibody molecule that includes amino acidsequences of Complementarity Determining Regions (CDRs; i.e., HCDR1,HCDR2, and HCDR3), and Heavy Framework Regions (HFRWs).

The CDRs are interspersed with regions that are more conserved, termedframework regions (“FRW”), Each LCVR and HCVR. is composed of three CDRsand four FRWs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FRW1, CDR1, FRW2, CDR2, FRW3, CDR3, FRW4. The threeCDRs of the light chain are referred to as “LCDR I, :LCDR2, and LCDR3”and the three CDRs of the I1(are referred to as “HCDR1, HCDR2, andHCDR3.” The CDRs contain most of the residues which form specificinteractions with the antigen. The numbering and positioning of CDRamino acid residues within the LCVR and HCVR regions is in accordancewith known conventions (e.g., Kabat (1991), Chothia (1987), and/or North(2011)). In different embodiments of the invention, the FRWs of theantibody may be identical to the germline sequences, or may be naturallyor artificially modified.

In certain embodiments, the anti-CD2O Ab for the methods and/or uses ofthe present invention is altered to increase or decrease the extent towhich the antibody is glycosylated. Addition or deletion ofglycosylation sites to an antibody may be conveniently accomplished byaltering the amino acid sequence such that one or more glycosylationsites is created or removed.

Unless indicated otherwise, when referring to an amino acid residue inan antibody by a number, the EU numbering system is used herein as it isconventionally used in the art (see, Kabat, et al., Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and :Human Services, NIII Publication No, 91-3242 (1991), forexample).

As used herein, the term “kit” refers to a package comprising at leasttwo separate containers, wherein a first container contains a K9LO-133Ab and a second container contains pharmaceutically acceptable carriers,diluents, or excipients. As used herein, the term “kit” also refers to apackage comprising at least two separate containers, wherein a firstcontainer contains K9LO-133 Ab, and another antibody preferably for thetreatment of cancers other than lymphomas. A “kit” may also includeinstructions to administer all or a portion of the contents of thesefirst and second containers to a cancer patient. Optionally, these kitsalso include a third container containing a composition comprising aknown chemotherapeutic agent.

As used herein, the terms “treating,” “to treat,” or “treatment” refersto restraining, slowing, stopping, reducing, or reversing theprogression or severity of an existing symptom, disorder, condition, ordisease.

As used herein, the term “effective amount” refers to the amount or doseof a. caninized anti-CD20 Ab which, upon single or multiple doseadministration to the patient, provides an effective response in thepatient under diagnosis or treatment.

As used herein, the terms “effective response” of a patient or apatient's “responsiveness” to treatment with a combination of agents, or“therapeutic effect” refers to the clinical or therapeutic benefit(s)imparted to a patient upon administration of a caninized anti-CD20 Ab.Such benefit(s) include any one or more of: extending survival(including overall survival and progression free survival); resulting inan objective response (including a complete response or a partialresponse); decreasing amount of B-cells, decreasing concentration ofB-cells in a patient's blood or other tissues and fluids tumorregression, tumor weight or size shrinkage, longer time to diseaseprogression, increased duration of survival, longer progression freesurvival, improved overall response rate, increased duration ofresponse, and improved quality of life and/or improving signs orsymptoms of cancer, etc.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount for a patient, a number of factors areconsidered by the attending diagnostician, including, but not limitedto: the species or breed of patient; its size, age, and general health;the specific disease or disorder involved; the degree of or involvementor the severity of the disease or disorder; the response of theindividual patient; the particular compound administered; the mode ofadministration;

the bioavailability characteristics of the preparation administered; thedose regimen selected; the use of concomitant medication; and otherrelevant circumstances.

K9LO-133

AME-133V is a second generation humanized IgG 1. monoclonal antibody.K9LO-133 is a partially caninized (human variable regions with canineconstant regions) isotype C monoclonal antibody version of AME-133V thatspecifically binds to CD20 protein in dogs.

In an embodiment, K9LO-133 contains engineered variable regions of theheavy and light chains, CDRs and frameworks. In an embodiment, a residuevariant in LCDR1 of K9LO-133 is introduced. In an embodiment, theresidue variant in LCDR1of K9LO-133 is V165G. In an embodiment, avariant hinge region is introduced. In an embodiment, a variant regiondownstream of the hinge is added. In an embodiment, the variant regiondownstream of the hinge allows K9LO-133 to bind to protein A.

K9LO-133 has a heavy chain amino acid sequence corresponding to SEQ IDNO: 1 and a corresponding nucleotide sequence SEQ ID NO: 2 that encodesfor the amino acid of SEQ ID NO: 1. K9LO-133 has a heavy chain variableregion sequence of SEQ ID NO: 3. The heavy chain variable sequenceregion further consists of HFWK 1 (SEQ ID NO: 4), HCDR1 (SEQ ID NO: 5),HFWK 2 (SEQ ID NO: 6), HCDR2 (SEQ ID NO: 7), HFWK3 (SEQ ID NO: 8), andHCDR3 (SEQ ID NO: 9).

K9LO-133 has a light chain amino acid sequence corresponding to SEQ IDNO: 10 and a corresponding nucleotide sequence SEQ ID NO: 11 thatencodes for the amino acid of SEQ ID NO: 10. K9LO-133 has a light chainvariable region sequence of SEQ ID NO: 12. The light chain variablesequence region further consists of LFWK 1 (SEQ ID NO: 13), LCDR1 (SEQID NO: 14), LFWK 2 (SEQ ID NO: 15), LCDR2 (SEQ ID NO: 16), LFWK 3 (SEQID NO: 17), LCDR3 (SEQ_(.) ID NO: 18), and a J region (SEQ ID NO: 19).

CD 2O Expression in Canine Lymphoma Tissue

CD 2O expression in canine lymphoma tissue can be evaluated usingimmunohistochemistry (IHC) with AME-133V and K9LO-133. In order todetect binding, AME-133V and K9LO-133 can be applied to cryosections ofcanine lymphoma samples at one concentration, for example10 μg/mL. Inaddition, AME-133V and K9LO-133 can be substituted with an appropriatespecies and isotype matched negative control antibody which has adifferent antigenic specificity from that of detection antibodies, forexample, either human IgG1, designated HuIgGl, (for AME-133V) and dogIgG, designated DgIgG (for

K9LO-133). Other controls can be produced by omission of the detectionantibodies or negative control antibodies from the assay (assaycontrol).

When tested essentially as described above, AME-133V and K9LO-133produced 3+ staining of >75% of the positive control mononuclearleucocytes. For K9LO-133, 3+ staining was observed in lymphocytes in thewhite pulp in canine spleen. AME-133′V and K9LO-133 did not specificallyreact with the negative control smooth myocytes (specifically nuclei forK9LO-133) in canine spleen. The negative control antibodies, HuIg,G1 andDgIgG, did not specifically react with either the positive or negativecontrol tissue elements in canine spleen. There also was no staining ofthe assay control slides. The specific reactions of AME-133V andK9LO-133 with the positive control tissue element and the lack ofspecific reactivity with the negative control tissue element, as well asthe lack of reactivity of the negative control antibody, indicated thatthe assay was sensitive, specific, and reproducible.

CD20 was detected in >75% of the neoplastic cells in 8 of 10 caninelymphoma samples examined. In the other samples, 1-5% of neoplasticcells were positively stained for CD20.

K9LO -133 Treated Dogs

The human dose for Rituximab for the treatment of non-Hodgkin's lymphomais 375 mg/m². Generally, a canine chemotherapeutic dose is 40% of ahuman dose, irrespective of cancer type. Therefore, and without beingbound by theory, an appropriate starting dose of an anti-CD20 antibodyfor canine non-Hodgkin's lymphoma is about 150 mg/m². In order to adjustfor body surface area of a 7 kg dog, the 150 mg/m² number was correctedby multiplying by 0.37. The resulting dose of K9LO-133 administered tothe dogs was about 57 mg/dog.

In an embodiment, the dose of K9LO-133 administered to a dog is fromabout 140 mg to about 160 mg per meter squared of body surface area. Inan embodiment, the dose of K9LO-133 administered to a dog is from about130 mg to about 170 mg per meter squared of body surface area. In anembodiment, the dose of K9LO-133 administered to a dog is from about 110mg to about 190 mg per meter squared of body surface area. In anembodiment, the dose of K9LO-133 administered to a dog is from about 50mg to about 250 mg per meter squared of body surface area.

In an embodiment, the dose of K9LO-133 administered to a dog is fromabout 50 mg/dog to about 60 mg/dog. In another embodiment, the dose ofK.9LO-133 administered to a dog is from about 40 mg/dog to about 70mg/dog. In an embodiment, the dose of K9LO-133 administered to a dog isfrom about 30 mg/dog to about 100 mg/dog. In an embodiment, the dose ofK9LO-133 administered to a dog is from about 10 mg/dog to about 1000mg/dog.

The efficacy of K.9LO-133 was determined in dogs by dosing themintravenously (IV) at about 150 mg/m² on Days 0, 7, and 35. Three femaleBeagle dogs were acclimated to study conditions for seven days duringwhich they were subjected to physical examinations; body weightmeasurements; and daily clinical observations. On dosing days_(;) dogswere dosed with 57 mg of K9L0-133 via slow IV infusion at a rate of 0.5to 1 mL/min.

Blood was collected for determination of lymphocyte proportions andserum retention on Days −5, 2, 9, 16, 23, 30, and 37.

As depicted in FIG. 1, there was a drop in the proportion of bloodB-lymphocytes after treatment with K9LO-133. The proportion ofB-lymphocytes (CD21+ and CD22+) dropped on Day 2, showed a recoverybetween Days 2 and 30, but had not yet returned to baseline levels byDay 37. CD8+ T-lymphocytes showed a decrease on Day 16 but had returnedto baseline levels by Day 23. The proportion of CD4+ T-lymphocytesremained relatively constant.

Thus, IV administration of K9LO-133 reduced B-lymphocyte counts incirculation with a transient decrease in CD8+ T-lymphocytes in treateddogs.

In an aspect, disclosed herein is an antibody having a light chain iableregion (LCVR) whose amino acid sequence is that given in SEQ ID NO: 12,and a heavy chain variable region (HCVR) whose amino acid sequence isthat given in SEQ ID NO: 3. In an embodiment, the antibody specificallybinds to canine CD20.

In another aspect, disclosed herein is an antibody having a light chain(LC) whose amino acid sequence is that given in SEQ ID NO: 10, and aheavy chain (HC) whose amino acid sequence is that given in SEQ ID NO: 1is disclosed. In an embodiment, the antibody specifically binds tocanine CD20.

In an aspect, disclosed herein is an antibody having two light chains(LC) each having an amino acid sequence that is given in SEQ ID NO: 10,and two heavy chains (HC) each having an amino acid sequence that isgiven in SEQ ID NO: 1, In an embodiment, the antibody specifically bindsto canine CD20.

In another aspect, a method of treating lymphoma in a canine patient isdisclosed that includes administering to a lymphoma cancer caninepatient in need of such treatment an effective amount of an antibodyhaving a light chain variable region (LCVR) whose amino acid sequence isthat given in SEQ ID NO: 12, and a heavy chain variable region (HCVR)whose amino acid sequence is that given in SEQ ID NO: 3, and theantibody specifically binds to canine CD20. In an embodiment, theantibody has a light chain (LC) whose amino acid sequence is that givenin SEQ ID NO: 10, and a heavy chain (HC) whose amino acid sequence isthat given in SEQ ID NO: 1. In an embodiment, the antibody is K9LO-133.

In an aspect, a kit is disclosed that has a pharmaceutical compositioncontaining K9LO -133 with one or more pharmaceutically acceptablecarriers, diluents, or excipients.

In another aspect, an antibody is disclosed having a light chain (LC)whose amino acid sequence is that given in SEQ ID NO: 10, and a heavychain (HC) whose amino acid sequence is that given in SEQ ID NO: 1, andthe antibody specifically binds to canine CD20, and the antibody bindsto protein A.

EXAMPLES K9LO-133

K9LO-133 can be supplied in 10 mM citrate, 150 mM NaCL, pH 6.5 at 26.3mg/mL. The vehicle can be physiological saline at 0.9% NaCI.

Dosing of K9LO-133 can be based on body surface area, which wasestimated for the dogs in this study to be 0.37 m². Each dog received150 mg/m² (57 mg) of K9LO-133 at each dose. Doses were prepared bydrawing up 57 mg (2.2 mL) of K9LO-133 and diluting in 27.8 mL ofphysiologic saline to bring the total volume to 30 mL for infusion.

Flow Cytometry

T-lymphocyte (CD4+ and CD8+) and B-lymphocyte (CD21+ and CD22+) amountscan be determined by flow cytometry. Blood collection for flow cytometrywas performed prior to feeding. Approximately 1 mL of EDTAanti-coagulated blood was provided from 3 dogs at baseline (day −5), andon Days 2, 9, 16, 23, 30 and 37. Red blood cells were lysed withammonium chloride hypotonic buffer, and leukocytes were stained withantibodies reactive with canine CD4, CD8, C,D21. and CD22. Then, unboundantibodies were removed and the acquisition of 10,000 leukocyte eventsin a FACScan or a FACSAria flow cytometer were obtained.

Lymphoma Samples

Ten frozen canine lymphoma samples were trimmed and embedded inTissue-Tek® OCT (Optimal Cutting Temperature) compound and stored in afreezer set to maintain −80 ° C. until sectioning. Sections were cut atapproximately 5 μm to generate an appropriate number of slides forsubsequent IHC staining.

Immunohistochemistry Staining Sections from each lymphoma sample werestained using AME-133V, a human IgG1 monoclonal antibody directedagainst human CD20, and K9LO-133, a chimeric canine isotype C monoclonalantibody directed against canine CD20, using IHC as detailed below.AME-133V was in a 19.5 mg/mL solution in phosphate-buffered saline(PBS), pH 7.4. K9LO-133 was in a 2.5 mg/mL solution in PBS, pH 7.4.

IHC staining was performed including use of positive and negativecontrol materials in each staining run and using additional slides forthe negative control antibody and assay control to ensure stainspecificity. The staining procedures, primary and secondary antibodydilutions, and controls for AME-133V were qualified in preliminarystaining runs. The staining procedures, primary and secondary antibodydilutions, and controls for K9LO-133 were qualified in a methodsdevelopment staining run.

AME-133V Staining

The indirect immunoperoxidase procedure can be used to stain the caninelymphoma tissue with AME-133V to detect CD20. Slides were fixed inacetone for 10 minutes at room temperature at the time of sectioning.Acetone-fixed ci osections were rinsed twice in phosphate-bufferedsaline, 0.15 M NaCl, pH 7.2 PBS. Endogenous peroxidase was then quenchedby incubation of the slides with Biocare PeroxAbolish for 5 minutes atroom temperature. Next, the slides were rinsed twice with PBS, incubatedwith the avidin solution for 15 minutes, rinsed once with PBS, incubatedwith the biotin solution for 15 minutes, and rinsed once with PBS. Theslides were then treated with a protein block designed to reducenonspecific binding for 2.0 minutes. The protein block was prepared asfollows: PBS+1% bovine serum albumin (BSA); 0.5% casein; and 3% donkeyserum.

Following the protein block, the primary antibodies AME-133V, HumanIgG1, or none were applied to the slides at a concentration of 10 μg/mLfor 1 hour. Then, the slides were rinsed twice with PBS, and thebiotinylated secondary antibody (donkey anti-human IgG) was applied tothe slides for 30 minutes. Next, the slides were rinsed twice with PBS,reacted for 30 minutes with the ABC Elite reagent, and rinsed twice withPBS. Next, DAB was applied for 4 minutes as a substrate for theperoxidase reaction. All slides were rinsed with tap water,counterstained, dehydrated, and mounted. PBS+1% BSA served as thediluent for primary antibodies and ABC Elite reagent. (PBS+1%BSA)+canine IgG (1:25 dilution) served as the diluent for the secondaryantibody.

K9-LO-133 Staining

The indirect immunoperoxidase procedure can be used to stain the caninelymphoma tissue with K9LO-133 to detect CD20. The requirement forlabeling (e.g. biotin, peroxidase, or fluorescein) of K9LO-133 andpreclusion of nonspecific reactivity between the secondary^(,) labeledanti-canine IgG and IgG endogenous to the tissues examined can beeliminated according to the following process. The labeled secondaryantibody was allowed to attach specifically to the unlabeled primaryantibody (K9LO-133, DgIgG, or none) by overnight incubation of theprimary/secondary antibody mixtures prior to application to the tissuecryosections. The detection reagent or negative control antibody (at aconcentration of 10 μg/mL) was mixed with biotinylated rabbit anti-dogIgG, Fc fragment-specific antibody (RbαDgIgG) at a concentration of 15μg/mL to achieve a primary:secondary antibody ratio of 1:1.5 on the dayprior to staining.

Precomplexed antibodies were incubated overnight on a rocker mechanismin a refrigerator set to maintain 4° C. Prior to use of the antibody onthe subsequent day, dog gamma globulins were added to each vial toachieve a final concentration of 3 mg/mL, and then incubated for atleast 2 hours on the rocker mechanism in a refrigerator set to maintain4° C. Slides were fixed in acetone for 10 minutes at room temperature atthe time of sectioning, On the day of staining, the slides were rinsedtwice with Tris-buffered saline, 0.15M NaCl, pH 7.6 TBS. Endogenousperoxidase was then quenched by incubation of the slides with anappropriate reagent such as Biocare PeroxAbolish reagent for 5 minutesat room temperature. Next, the slides were rinsed twice with TBS,incubated with the avidin solution for 15 minutes, rinsed once with TBS,incubated with the biotin solution for 15 minutes, and rinsed once withTBS. The slides were then treated with a protein block designed toreduce nonspecific binding for 20 minutes.

The protein block was prepared as follows: TBS+1% bovine serum albumin(BSA);

0.5% casein; and 3% normal rabbit serum. Following the protein block,the precomplexed primary and secondary antibodies were applied to theslides for two hours. Next, the slides were rinsed twice with TBS,treated with the ABC Elite reagent for 30 minutes, rinsed twice withTBS, and then treated with DAB for 4 minutes as a substrate for theperoxidase reaction. All slides were rinsed with tap water,counterstained, dehydrated, and mounted. TBS -1- 1%

BSA served as the diluent for all antibodies and ABC reagent. Eachstaining run included a positive control (mononuclear leucocytes), and anegative control (smooth myocytes) of cryosections of canine spleen.

SEQUENCE LISTING PRT; Artificial Sequence SEQ ID NO: 1EVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAIYPLTGDTSYNQKSKLQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYVGGDWQFDVWGKGTTVTVSSASTTAPSVFPLAPSCGSQSGSTVALACLVSGYIPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPATNTKVDKPVPKRENGRVPRPPDCPKCPAPELLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGIGHQDWLSGKQFKCKVNNKALPSPIEKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGK; DNA; Artificial SequenceSEQ ID NO: 2 GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGCCGTACATTTACCAGTTACAATATGCACTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGGCTATTTATCCCTTGACGGGTGATACTTCCTACAATCAGAAGTCGAAACTCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGATCGACTTACGTGGGCGGTGACTGGCAGTTCGATGTCTGGGGCAAGGGGACCACGGTCACCGTCTCCTCAGCCTCCACCACGGCCCCCTCGGTTTTCCCGCTAGCGCCCAGCTGTGGGTCCCAATCCGGCTCCACGGTGGCCCTGGCCTGCCTGGTGTCAGGCTACATCCCCGAGCCTGTAACTGTGTCCTGGAATTCCGGCTCCTTGACCAGCGGTGTGCACACCTTCCCGTCCGTCCTGCAGTCCTCAGGGCTCTACTCCCTCAGCAGCATGGTGACAGTGCCCTCCAGCAGGTGGCCCAGCGAGACCTTCACCTGAATGTGGCCCACCCGGCCACCAACACTAAAGTAGACAAGCCAGTGCCCAAAAGAGAAAATGGAAGAGTTCCTCGCCCACCTGATTGTCCCAAATGCCCAGCCCCTGAACTGCTGGGAGGGCCTTCGGTCTTCATCTTTCCCCCAAAACCCAAGGACACCCTCTTGATTGCCCGAACACCTGAGGTCACATGTGTGGTGGTGGATCTGGACCCAGAAAACCCTGAGGTGCAGATCAGCTGGTTCGTGGATAGTAAGCAGGTGCAAACAGCCAACACGCAGCCTCGTGAGGAGCAGTCCAATGGCACCTACCGTGTGGTCAGTGTCCTCCCCATTGGGCACCAGGACTGGCTTTCAGGGAAGCAGTTCAAGTGCAAAGTCAACAACAAAGCCCTCCCATCCCCCATTGAGGAGATCATCTCCAAGACCCCAGGGCAGGCCCATCAGCCTAATGTGTATGTCCTGCCGCCATCGCGGGATGAGATGAGCAAGAATACGGTCACCCTGACCTGTCTGGTCAAAGACTTCTTCCCACCTGAGATTGATGTGGAGTGGCAGAGCAATGGACAGCAGGAGCCTGAGAGCAAGTACCGCATGACCCCGCCCCAGCTGGATGAAGATGGGTCCTACTTCCTATACAGCAAGCTCTCCGTGGACAAGAGCCGCTGGCAGCGGGGAGACACCTTCATATGTGCGGTGATGCATGAAGCTCTACACAACCACTACACACAGATATCCCTCTCCCATTCTCCGGGTAAATGATGATAG;PRT; Artificial Sequence SEQ ID NO: 3EVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAIYPLTGDTSYNQKSKLQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYV GGDWQTDVWGKGTTVTVSS;PRT; Artificial Sequence SEQ ID NO: 4 EVQLVQSGAEVKKPGESLKISC;PRT; Artificial Sequence SEQ ID NO: 5 KGSRTFTSYNMH;PRT; Artificial Sequence SEQ ID NO: 6 WVRQMPGKGLEWMG;PRT; Artificial Sequence SEQ ID NO: 7 AIYPLTGDTSYNQKSKL;PRT; Artificial Sequence SEQ ID NO; 8 QVTISADKSISTAYLQWSSLKASDTAMYYC;PRT; Artificial Sequence SEQ ID NO: 9 ARSTYVGGDWQFDV;PRT; Artificial Sequence SEQ ID NO: 10EIVLTQSPGTLSLSPGERATLSCRASRSVPYIHWYQQKPGQAPRLLIYATSALASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWLSNPPTFGQGTKLEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLI KSFQRSECQRVD;DNA; Artificial Sequence SEQ ID NO; 11GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGCCGGAGTGTACCGTACATCCACTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGCCACATCCGCTCTGGCTTCTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTGGCTGAGTAACCCACCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAAATGATGCCCAGCCAGCCGTCTATTTGTTCCAACCATCTCCAGACCAGTTACACACAGGAAGTGCCTCTGTTGTGTGCTTGCTGAATAGCTTCTACCCCAAAGACATCAATGTCAAGTGGAAAGTGGATGGTGTCATCCAAGACACAGGCATCCAGGAAAGTGTCACAGAGCAGGACAAGGACAGTACCTACAGCCTCAGCAGCACCCTGACGATGTCCAGTACTGAGTACCTAAGTCATGAGTTGTACTCCTGTGAGATCACTCACAAGAGCCTGCCCTCCACCCTCATCAAGAGCTTCCAAAGGAGCGAGTGTCAGAGAGTGGAC; PRT; Artificial SequenceSEQ ID NO: 12 EIVLTQSPGTLSLSPGERATLSCRASRSVPYTHWYQQKPGQAPRLLIYATSALASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWLSNPPTFGQGTK LEIK;PRT; Artificial Sequence SEQ ID NO: 13 EIVLTQSPGTLSLSPGERATLSC;PRT; Artificial Sequence SEQ ID NO: 14 RASRSVPYIH;PRT; Artificial Sequence SEQ ID NO; 15 WYQQKPGQAPRLLI;PRT; Artificial Sequence SEQ ID NO: 16 YATSALAS;PRT; Artificial Sequence SEQ ID NO: 17 GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC;PRT; Artificial Sequence SEQ ID NO; 18 QQWLSNPPT;PRT; Artificial Sequence SEQ ID NO: 19 FGQGTKLEIK;

We claim:
 1. An antibody comprising a light chain variable region (LCVR)whose amino acid sequence is that given in SEQ :ID NO: 12, and a heavychain variable region (HCVR) whose amino acid sequence is that given inSEQ ID NO:
 3. 2. The antibody of claim 1, wherein the antibodyspecifically binds to canine CD20.
 3. An antibody comprising a lightchain (LC) whose amino acid sequence is that given in SEQ ID No: 10, anda heavy chain (HC) whose amino acid sequence is that given in SEQ IDNO:
 1. 4. The antibody of claim 3, wherein the antibody specificallybinds to canine CD20.
 5. An antibody comprising two light chains (LC)each having an amino acid sequence that is given in SEQ ID NO: 10, andtwo heavy chains (HC) each having an amino acid sequence that is givenin SEQ ID NO:
 1. 6. The antibody of claim 5, wherein the antibodyspecifically binds to canine CD20.
 7. A method of treating lymphoma in acanine patient, comprising administering to a lymphoma cancer caninepatient in need of such treatment an effective amount of an antibodycomprising a light chain variable region (LCVR) whose amino acidsequence is that given in SEQ ID NO: 12, and a heavy chain variableregion (HCVR) whose amino acid sequence is that given in SEQ ID NO: 3,wherein the antibody specifically binds to canine CD20.
 8. The methodaccording to claim 7 wherein the antibody comprises a light chain (LC)whose amino acid sequence is that given in SEQ ID NO: 10, and a heavychain (HC) whose amino acid sequence is that given in SEQ ID NO:
 1. 9.The method according to claim 7, wherein the antibody is K9LO-133.
 10. Akit, comprising a pharmaceutical composition, comprising K9LO-133, withone or more pharmaceutically acceptable carriers, diluents, orexcipients.
 11. An antibody comprising a light chain (LC) whose aminoacid sequence is that given in SEQ ID NO: 10, and a heavy chain (HC)whose amino acid sequence is that given in SEQ :11) NO: 1 and whereinthe antibody specifically binds to canine CD20, and wherein the antibodybinds to protein A.
 12. Use of an antibody comprising a light chainvariable region (LCVR) whose amino acid sequence is that given in SEQ IDNO: 12, and a heavy chain variable region (HCVR) whose amino acidsequence is that given in SEQ ID NO: 3, wherein the antibodyspecifically binds to canine CD20, for treating lymphoma in a caninepatient.
 13. The use according to claim 12 wherein the antibodycomprises a light chain (LC) whose amino acid sequence is that given inSEQ ID NC): 10, and a heavy chain (HC) whose amino acid sequence is thatgiven in SEQ ID NO:
 1. 14. The use according to claim 12, wherein theantibody is K9LO-133.
 15. An antibody comprising a light chain variableregion (LCVR) whose amino acid sequence is that given in SEQ ID NO: 12,and a heavy chain variable region (HCVR) whose amino acid sequence isthat given in SEQ ID NO: 3, wherein the antibody specifically binds tocanine CD20, for use in treating lymphoma in a canine patient.